Post-transfusion hepatitis refers to hepatitis caused by transfusion as the name implies, and hepatitis B virus (HBV) is the first one identified as the causative virus of post-transfusion hepatitis. In HBV antigen testing, methods for detecting hepatitis B virus surface (HBs) antigen have been used in blood screening, and methods for detecting hepatitis B virus e (HBe) antigen have been commonly used as a marker for the replication of hepatitis B virus.
HBe antigen is a pre-core protein expressed by the same promoter as that for hepatitis B virus core protein (HBc antigen) constituting the HBV particle. Since this protein is aggressively produced and secreted during the replication of HBV, the amount of HBe antigen in the blood is thought to largely reflect the amount of HBV when HBe antibody is absent. However, once the production of HBe antibody is initiated, HBe antigen forms immune complex leading to the establishment of seroconversion in which HBe antibody can only be detected. In such specimens, no HBe antigen is detected and the amount of HBV is not reflected.
On the other hand, cases have been reported in which, even at the state of seroconversion indicating the quiescence of type B hepatitis, levels of alanine aminotransferase (ALT), an indicator of hepatitis activity, may vary, and since HBV DNA was detected by the polymerase chain reaction (PCR), the presence of a precore mutant was confirmed. Precore mutants mean that since a codon at position 28 of the prepro HBe protein was mutated to a stop codon, HBe antigen can no longer been produced or secreted with a result that HBe antigen has become negative. In other words, it became clear that the measurement of HBe antigen and antibody alone is not sufficient for monitoring HBV carriers.
With the spread of the nucleic acid amplification tests (NATs), attention has been given to the relationship between the amount of HBV DNA and the pathology of HBV carriers, and accordingly NATs have been primarily used for monitoring after medication with anti-viral agents.
Though nucleic acid amplification tests such as the PCR method and the TMA method are highly sensitive methods for detecting gene fragments, however, they are complicated in that they require two hours of hands-on time in extracting HBV genomic DNA from test samples in the manual method, and involve several steps of procedures. In addition, such complicated procedures increase chances of contamination, and thereby increased possibilities of false positive samples. Furthermore, technical skills are required in order to obtain quantitative values in a stable manner, and utmost attention has to be paid in the storage of test samples in order to detect biochemically unstable substances such as DNA. This makes it hard to process a large quantity of samples at one time. Though contamination measures have been improved and processing time for DNA extraction has been curtailed in recent years due to the development of automated equipment, expensive instruments are still required, and accordingly the method has not been generally used except in facilities that process a large amount of samples. Furthermore, since the DNA primer must be in agreement with the target gene, several types of primers must be used, which poses a problem since cost per test becomes higher as compared to immunoassays.
In stead of the above methods that detect the HBV genome, methods of directly detecting HBV core antigen (HBc antigen) have been developed. Usuda et al. (Journal of Virological Methods, 72:95-103, 1998) have developed a method for detecting HBc antigen in the serum using monoclonal antibody that has specificity for HBV core (HBc) antigen, and demonstrated that it has clinical usefulness similarly to the above NAT tests that detect the viral genome. This method, however, still has problems in several points.
First, when compared to the NAT method, it is less sensitive with a detection limit of 105 copies/ml in terms of the amount of HBV DNA, and therefore can not be used in serum screening or monitoring tests.
Besides, steps of processing samples for measurement are complicated and are time-consuming, which poses problems when it is to be used in applications such as screening and monitoring. Thus, for the processing of test samples (sera), multi-stage processing is required for the concentration of viral particles and the removal of serum components, including treatment with HBs polyclonal antibody (37° C., two hours), centrifugation procedure (10 minutes), supernatant removal, treatment with surfactants, alkali treatment (35 minutes), and the addition of neutralizing agents. Such processes require highly experienced technical skills, and in order to attain reproducibility, trained skills and a processing time of at least three hours are required. Furthermore, due to steps of centrifugation, supernatant removal etc., the method is refractory to automation, and makes simultaneous bulk handling difficult, and therefore it is not suitable for applications that require bulk handling from the viewpoint of processing.
Because of these problems, it has not been put into practical use in laboratory testing.
On the other hand, the HBc antigen detection system has advantages over the NAT method in the following points. Thus, since the detection process is not accompanied by an amplification procedure, it relatively tolerates contamination. Furthermore, since it detects antigen protein which is relatively stable in stead of biochemically unstable substances such as DNA, excessive care need not be taken on the storage of test samples, which permits easier transport thereof.
These features are important requirements in applications where a large number of test samples are measured as in the blood business and physical checkups. However, the disclosed Method for detecting HBc antigen is not suitable for automation because of complicated pretreatment, and cannot be used in screening or therapeutic monitoring because of low sensitivity, and therefore the method has not utilized the advantages over the NAT method to the full extent. In addition, clinically useful methods of measurement must always address the problems of sensitivity, specificity, reproducibility, ease of operation and low cost, and must be intensively developed so as to satisfy all of these.
Literature so far reports antibodies that recognize the sequence region of the amino acid Nos. shown in [ ] on HBc antigen.
[73-89]; A. Semiletov Iu et al., Bioorg Khim 20 (11), 1175-85 (1994)
[124-133], [135-147]; M. Sallberg et al., J Gen Virol 74 (Pt7), 1335-40 (1993)
[N terminal], [134-140]; V. Skrivelis et al., Scand J Immunol 37 (6), 637-43 (1993)
[2-10], [134-140], [138-154]; V. Bichko et al., Mol Immunol 30(3), 221-31 (1993)
[126-135]; M. Sallberg et al., Mol Immunol 28(7), 719-26 (1991)
[76-85]; M. Sallberg et al., J Med Virol 33(4), 248-52 (1991)
[73-85], [107-118]; G. Colucci et al., J Immunol 141(12), 4376-80 (1988)
[9-20], [78-83], [127-133], [133-145]; P. Pushko et al., Virology 202(2), 912-20 (1994)
By using these antibodies in combination with a method of pretreating test samples, it is possible to make up a system for measuring HBc antigen. However, highly sensitive and highly specific systems of measurement have not been established yet.